Compositions And Methods For Targeting Connexin Hemichannels

ABSTRACT

Methods for identifying compounds that positively regulate connexin 43 hemichannels.

PRIORITY PARAGRAPH

This application is an international application claiming priority toU.S. Provisional Application Ser. No. 61/868,112 filed Aug. 21, 2013,which is incorporated herein by reference in its entirety.

BACKGROUND

Bone tissues are a preferred site of breast and prostate cancermetastasis. Bone metastasis occurs in up to 75% of patients withadvanced cancers. Currently, there is no cure for metastatic breastcancer and no reliable intervention drug for treating bone metastasisthat has minimal side effects.

Osteoarthritis (OA) is a prevalent disease that affects approximately20% of U.S. adults. This disease causes the degeneration of jointsincluding articular cartilage and subchondral bone. The pathology of OAis characterized by a loss of articular cartilage leading to narrowingof joint space, increased joint friction and potential structureremodeling. Current treatment includes exercise, lifestyle change andanalgesics. If symptom becomes severe, joint replacement surgery isnormally performed. Thus far, there is no specific pharmaceuticalintervention available for the treatment of OA.

Connexin hemichannels play important roles in the cell and tissuefunction, and abnormal function of connexin hemichannels is known tocause various pathological conditions. Thus, there remains a need foradditional therapies for treating pathological conditions associatedwith hemichannels activity (e.g., inflammation, osteoarthritis, or bonemetastasis), as well as methods for identifying such therapies.

SUMMARY

The inventors have discovered that open hemichannels in cells ofmetastatic targets have inhibitory effects on cancer growth, migration,and metastasis. Certain embodiments provide a tool and/or method toidentify compounds that modulate opening of hemichannels for thetreatment of cancer metastasis. In certain aspects the drugs can be usedto inhibit or ameliorate cancer metastasis to the bone, brain, or liver.In certain aspects a hemichannel can be expressed in a bone, brain, orliver cell. In a further aspect the hemichannel can be an osteocytehemichannel, a hepatocyte hemichannel, or an astrocyte hemichannel. Incertain aspects a hemichannel can be a connexin Cx43, Cx32, Cx46, Cx37,Cx40, Cx50, Cx59, Cx62, Cx26, Cx31, Cx30.3, Cx31.1, Cx30, Cx25, Cx45,Cx47, Cx30.2, Cx36, Cx31.9, Cx39, Cx40.1, Cx23, or Cx29 hemichannel. Incertain aspect the hemichannel is a Cx43 or Cx 32 hemichannel. As anexample, opening of connexin 43 (Cx43) hemichannels in osteocytes has aninhibitory effect on metastasis to the bone and can suppress bonemetastasis.

Hemichannel opening can be detected by dye uptake assays usingfluorescence dyes like Lucifer yellow, ethidium bromide, Alexa 350,Alexa 485, Alexa 594 dyes, etc. The specificity of the hemichannelopening can be verified by using a connexin specific antibody thatinhibits hemichannel opening and thus inhibits the activity of thetarget reagent. Therefore, the tools and/or methods described can beused for screening, testing, and identifying reagent(s) that openhemichannels and inhibit metastasis.

Certain embodiments are directed to methods of identifying a compoundthat open hemichannels. Other embodiments are directed to methods ofpositively modulating the opening of hemichannels in to inhibit orameliorate cancer metastasis.

The present invention provides antibodies directed against ahemichannel, nucleic acids encoding such antibodies and therapeuticproteins, methods for preparing anti-hemichannel monoclonal antibodiesand other therapeutic proteins, and methods for the treatment ofdiseases, such as metastatic cancer. In certain aspects the antibodybinds an epitope having an amino acid sequence of FLSRPTEKTI (SEQ ID NO:13), KRDPCPHQVD (SEQ ID NO:14), or LSAVYTCKR (SEQ ID NO:15). In aparticular aspect an antibody binds an epitope having an amino acidsequence of FLSRPTEKTI (SEQ ID NO: 13).

In one embodiment, the invention provides an isolated antibody whichspecifically binds to hemichannels, comprising a heavy chain having anamino acid sequence of SEQ ID NO:2 and a light chain having an aminoacid sequence of SEQ ID NO:4.

In certain aspects a first heavy chain region comprises an amino acidsequence having an amino acid sequence of residues 13 to 37 of SEQ IDNO:2; a second heavy chain region having an amino acid sequencecorresponding to residues 46 to 66 of SEQ ID NO:2; and a third heavychain region comprising an amino acid sequence having an amino acidsequence of residues 97 to 116 of SEQ ID NO:2.

In another aspect a first light chain region comprises an amino acidsequence having an amino acid sequence of residues 9 to 40 of SEQ IDNO:4; a second light chain region having an amino acid sequencecorresponding to residues 49 to 58 of SEQ ID NO:4; and a third lightchain region comprising an amino acid sequence having an amino acidsequence of residues 64 to 108 of SEQ ID NO:4.

In one embodiment, the invention provides an isolated antibody whichspecifically binds to hemichannels and gap junctions, comprising a heavychain having an amino acid sequence of SEQ ID NO:6 and a light chainhaving an amino acid sequence of SEQ ID NO:8.

In certain aspects a first heavy chain region comprises an amino acidsequence having an amino acid sequence of residues 13 to 37 of SEQ IDNO:6; a second heavy chain region having an amino acid sequencecorresponding to residues 46 to 66 of SEQ ID NO:6; and a third heavychain region comprising an amino acid sequence having an amino acidsequence of residues 97 to 116 of SEQ ID NO:6.

In another aspect a first light chain region comprises an amino acidsequence having an amino acid sequence of residues 9 to 42 of SEQ IDNO:8; a second light chain region having an amino acid sequencecorresponding to residues 51 to 60 of SEQ ID NO:8; and a third lightchain region comprising an amino acid sequence having an amino acidsequence of residues 66 to 125 of SEQ ID NO:8.

In one embodiment, the invention provides an isolated antibody whichspecifically binds to gap junctions, comprising a heavy chain having anamino acid sequence of SEQ ID NO: 10 and a light chain having an aminoacid sequence of SEQ ID NO: 12.

In certain aspects a first heavy chain region comprises an amino acidsequence having an amino acid sequence of residues 10 to 34 of SEQ IDNO: 10; a second heavy chain region having an amino acid sequencecorresponding to residues 43 to 59 of SEQ ID NO: 10; and a third heavychain region comprising an amino acid sequence having an amino acidsequence of residues 94 to 109 of SEQ ID NO: 10.

In another aspect a first light chain region comprises an amino acidsequence having an amino acid sequence of residues 9 to 40 of SEQ ID NO:12; a second light chain region having an amino acid sequencecorresponding to residues 49 to 58 of SEQ ID NO: 12; and a third lightchain region comprising an amino acid sequence having an amino acidsequence of residues 64 to 108 of SEQ ID NO: 12.

In certain aspects antibodies include full length antibodies, antibodyfragments, single chain antibodies, bispecific antibodies, minibodies,domain antibodies, synthetic antibodies and antibody fusions, andfragments thereof.

A further embodiment provides a pharmaceutical composition comprising anantibody as described herein with a pharmaceutically acceptable carrier.Also provided is an antibody or a pharmaceutical composition of theinvention for use as a medicament or for use in therapy for cancer andto inhibit cancer metastasis.

A further embodiment provides a method of treating or preventing cancermetastasis. A method of treating can comprise administering to a subjectin need thereof an effective amount of an isolated antibody describedherein. Also provided is the use of an antibody as described herein inthe manufacture of a medicament for the treatment or prevention ofcancer metastasis.

Certain aspects are directed to in vitro methods of using an antibody,compounds or reagents to suppress inflamatory reactions in chondrocytes.In certain aspects methods are directed to determining the effect oninhibition of Cx43 hemichannel opening in chondrocytes by (i)determining hemichannel opening by dye uptake assay, using Luciferyellow or Alexa dyes, (ii) assessing inhibitory effects on hemichannelsopening by IL-1β, (iii) test inhibitory effects of the reagents onhemichannels opening by mechanical loading in the form of fluid flowshear stress.

Certain aspects are directed to methods of determining the effect of anantibody, compounds or reagents on suppressing of inflammatory responsesevoked by IL-1β and mechanical loading by (i) determining the inhibitionof activation of nuclear factor-kappaB (NF-κB) induced by IL-1β, (ii)determining the inhibition of activation of NF-κB induced by fluid flowshear stress.

Other aspects are directed to an in vivo method of using a monoclonalantibody, compounds or reagents to treat OA or identify the samecomprising (i) injecting antibody, compound or reagent into knee capcavity, (ii) assessing the inhibition of activation of NF-κB induced byIL-1β, (iii) assessing OA development by X-ray, histological analysisand physical movement.

As used herein, the term “antigen” is a molecule capable of being boundby an antibody or T-cell receptor. In certain embodiments, bindingmoieties other than antibodies and be engineered to specifically bind toan antigen, e.g., aptamers, avimers, and the like.

The term “antibody” or “immunoglobulin” is used to include intactantibodies and binding fragments/segments thereof. Typically, fragmentscompete with the intact antibody from which they were derived forspecific binding to an antigen. Fragments include separate heavy chains,light chains, Fab, Fab′ F(ab′)2, Fabc, and Fv. Fragments/segments areproduced by recombinant DNA techniques, or by enzymatic or chemicalseparation of intact immunoglobulins. The term “antibody” also includesone or more immunoglobulin chains that are chemically conjugated to, orexpressed as, fusion proteins with other proteins. The term “antibody”also includes bispecific antibodies. A bispecific or bifunctionalantibody is an artificial hybrid antibody having two differentheavy/light chain pairs and two different binding sites. Bispecificantibodies can be produced by a variety of methods including fusion ofhybridomas or linking of Fab′ fragments. See, e.g., Songsivilai andLachmann, Clin Exp Immunol 79:315-21, 1990; Kostelny et al., J. Immunol.148:1547-53, 1992.

The term “isolated” can refer to a nucleic acid or polypeptide that issubstantially free of cellular material, bacterial material, viralmaterial, or culture medium (when produced by recombinant DNAtechniques) of their source of origin, or chemical precursors or otherchemicals (when chemically synthesized). Moreover, an isolated compoundrefers to one that can be administered to a subject as an isolatedcompound; in other words, the compound may not simply be considered“isolated” if it is adhered to a column or embedded in an agarose gel.Moreover, an “isolated nucleic acid fragment” or “isolated peptide” is anucleic acid or protein fragment that is not naturally occurring as afragment and/or is not typically in the functional state.

Moieties of the invention, such as polypeptides, peptides, antigens, orimmunogens, may be conjugated or linked covalently or noncovalently toother moieties such as adjuvants, proteins, peptides, supports,fluorescence moieties, or labels. The term “conjugate” or“immunoconjugate” is broadly used to define the operative association ofone moiety with another agent and is not intended to refer solely to anytype of operative association, and is particularly not limited tochemical “conjugation.”

The term “providing” is used according to its ordinary meaning “tosupply or furnish for use.” In some embodiments, the protein is provideddirectly by administering the protein, while in other embodiments, theprotein is effectively provided by administering a nucleic acid thatencodes the protein. In certain aspects the invention contemplatescompositions comprising various combinations of nucleic acid, antigens,peptides, and/or epitopes.

The phrase “specifically binds” or “specifically immunoreactive” to atarget refers to a binding reaction that is determinative of thepresence of the molecule in the presence of a heterogeneous populationof other biologics. Thus, under designated immunoassay conditions, aspecified molecule binds preferentially to a particular target and doesnot bind in a significant amount to other biologics present in thesample. Specific binding of an antibody to a target under suchconditions requires the antibody be selected for its specificity to thetarget. A variety of immunoassay formats may be used to selectantibodies specifically immunoreactive with a particular protein. Forexample, solid-phase ELISA immunoassays are routinely used to selectmonoclonal antibodies specifically immunoreactive with a protein. See,e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Press, 1988, for a description of immunoassay formats andconditions that can be used to determine specific immunoreactivity.

Other embodiments of the invention are discussed throughout thisapplication. Any embodiment discussed with respect to one aspect of theinvention applies to other aspects of the invention as well and viceversa. Each embodiment described herein is understood to be embodimentsof the invention that are applicable to all aspects of the invention. Itis contemplated that any embodiment discussed herein can be implementedwith respect to any method or composition of the invention, and viceversa. Furthermore, compositions and kits of the invention can be usedto achieve methods of the invention.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

Throughout this application, the term “about” is used to indicate that avalue includes the standard deviation of error for the device or methodbeing employed to determine the value.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.”

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofthe specification embodiments presented herein.

FIG. 1 illustrates one embodiment of a Fluid Flow Loop Apparatus.

FIG. 2 illustrates results of treating MLO-Y4 osteocytic cells with 20μM AD for 30 min in the absence or presence of 1 μg/ml Cx43(E2)antibody. Ethidium bromide dye uptake was conducted and quantified ascompared to non-treated basal level of uptake. Study was carried out incalcium conditions. Low calcium conditions were used as control (openshemichannels). Also illustrated are results of treating MLO-A5osteoblasts with AD treatment or AD and Cx43(E2) antibody.

FIG. 3 illustrates a model system to study the role of osteocytic Cx43hemichannels in mediating the effect of AD on cancer cell migration.Cx43 hemichannels in osteocytes are open by AD or FFSS. The releasedfactor(s) in the AD- or FFSS-treated CM decreases cancer cell migration.Cancer cells treated with control CM exhibit normal migration.

FIGS. 4A and 4B illustrate studies using a soft agaroseanchorage-independent growth assay, which is different fromanchorage-dependent growth. Only cancer cells can grow on soft agar andtheir growth on this matrix indicates the extent of the cancer cellproliferation. CM-AD from osteocytes decreases MDA-MB-231 colonyformation.

FIGS. 5A and 5B illustrate the results of studies using a wound healingmigration assay. CM-AD from osteocytes inhibits MDA-MB-231 cellmigration.

FIG. 6 illustrates results of another wound healing migration assay. ADdoes not have a direct effect on the migration of MDA-MB-231 cells.

FIGS. 7A and 7B illustrate results from a transwell migration assay. (B)Dots represent the cells that have migrated to the opposite end of theinsert and have been stained. The smaller dots are the pores throughwhich they migrate. In this assay protein A was used to remove the E2 Abfrom CM, and this causes the migration effect to be the same before E2Ab was added.

FIG. 8 illustrates results from another transwell migration assay.MDA-MB-231 migration is not affected by CM-AD from MLO-A5 osteoblasts.

FIG. 9 illustrates results from another transwell migration assay.MDA-MB-231 migration is decreased with CM from osteocytes stimulated bymechanical loading.

FIGS. 10A and 10B illustrate results from Py8119 breast cancer cellinjection into Cx43 conditional knockout (cKO) mice. Py8199 tumor growthand metastasis is increased in Cx43 cKO mice. (B) Tumor spread to othertissues in Cx43 cKO mice.

FIG. 11 illustrates results from Py8119 injection into Cx43 cKO mice.Py8119 tumor growth is increased in Cx43 cKO mice.

FIG. 12 illustrates immunolabeling of Cx43 by the three mAbs. OsteocyteMLO-Y4 cells were fixed with 70% ethanol at −20° C. for 20 min, blockedovernight, incubated with mAbs for 3 h RT at the concentrations shownabove. Goat anti-mouse FITC secondary antibodies were used to assess theactivities of these mAbs. WGA as a cell marker was labeled in red.

FIG. 13 shows hybridoma supernatants affinity purified by passingthrough Cx43 E2 column at pH 7.4. The western blots were performed with1:100 dilution of each monoclonal antibody. Polyclonal Cx43 Ab dilutionwas 1:300.

FIG. 14 shows that M1 blocks hemichannels, but not gap junctions; M2,blocks gap junctions, but not hemichannels; and M3 blocks both. (A)MLO-Y4 cells were incubated with media with low Ca²⁺ and Mg²⁺, acondition that induces the opening of Cx43 hemichannels. Dye uptakeassay was performed in the presence of EtBr with or without mAbs for 20min. (B) HeLa cells transfected with Cx43 were incubated with mAbs for 3h and a signal cell was microinjected with AlexaFluor 488 to evaluatethe extent of dye transfer assayed for gap junctions.

FIG. 15 illustrates parachuting dye transfer assay showing M2 and M7,but not M1 blocks gap junction channels. Hela cells transfected withCx43 was used for parachuting dye transfer experiments. Donor cells wereincubated with 5 μM calcein red-orange-AM (790 Da) which is permeable togap junctions and 5 μM Oregon green 488 BAPTA-2-AM (1752 Da) which isnon-permeable to gap junctions for 40 minutes at 37° C. Donor cells weretreated with trypsin and separated preloaded cells were layered(‘parachuted’) over the top of the unlabeled recipient cells at a 1:4donor to receiver ratio. Cells were allowed to attach for 90 min. Thecells were examined under a fluorescence microscope.

FIG. 16. Shows blockage of hemichannel opening induced by mechanicalloading by M1 and M7, but not M2. MLO-Y4 cells were pretreated with orwithout mAbs for 20 min. The cells were subjected to fluid flow shearstress at 8 dynes/cm² for 10 min and dye uptake was performed with 100μM EtBr for 5 min. Cells were rinsed, fixed and images were taken underfluorescence microscopy.

FIG. 17. Shows that M1 blocks hemichannel opening induced by mechanicalloading in mouse bone osteocytes in vivo. Mouse IgG or Cx43(M1) mAb (25mg/kg) was injected 2 hr before Evans blue dye injection Into WT andCx43 cKO mice. 30 min after dye injection, left (L) tibias weremechanically loaded once for 10 min. Mice were euthanized and perfusedwith 50 ml PBS. Tibias were isolated and fixed and bone tissue sectionswere prepared. Bar, 40 mm. The arrowheads indicate dye uptake.

FIG. 18. Shows expression of Cx43 on the surface of chondrocytes. (A)Expression of Cx43 on the surface of primary chondrocytes. (B) Fluidflow shear (16 dynes/cm2) (FSS) opened hemichannels and this opening wassignificantly blocked by Cx43 specific antibody. FSS compared to allother conditions, ***, P<0.001.

DESCRIPTION

Various cells are able to communicate with each other and with theextracellular environment through hemichannels and gap junctions formedby the protein connexin. Connexin proteins are ubiquitously expressedthroughout the body. Six connexin proteins make up one hemichannel, and2 hemichannels make up 1 gap junction channel. Gap junctions are acluster of channels that are located in the plasma membrane betweenadjoining cells and they mediate intercellular communication.Hemichannels are a separate entity from gap junction channels.Hemichannels permit the exchange of molecules between the intracellularcompartments and the extracellular environment.

Osteocytes express hemichannels known as connexin (Cx) 43 hemichannels.These osteocyte hemichannels are normally closed and can be opened whenexposed to mechano-stimulation, which leads to the release of variousfactors into the bone microenvironment. The factors released byhemichannel opening can mediate other processes that can decrease tumorcell migration and bone metastasis.

Certain embodiments are directed to methods of identifying reagents thatmodulate the opening of connexin hemichannels. In certain aspects themethods identify compounds or drugs that positively modulate the openingof connexin hemichannels. Other embodiments are directed to methods oftreating cancer by administering a compound that open hemichannels to apatient having cancer. In certain aspects the patient has a primarytumor. In certain aspects compounds that open Cx43 hemichannels can beused to inhibit or reduce metastasis to the bone.

Cancer metastasis occurs when a cancer spreads from the part of the bodywhere it originated (e.g., breast or prostate) to other parts of thebody (e.g., liver or bone) and establishes a secondary tumor. The boneis one of the most common sites of cancer metastasis. Cancers thatmetastasize to bone include, but are not limited to breast cancer,prostate cancer, lung cancer, and skin cancers (e.g., melanoma). Bonemetastasis can be identified in up to 75% of patients with advancedbreast and prostate cancers. Bone metastasis (mets) are associated withmany significant clinical and quality of life consequences, such as, butnot limited to intractable pain, pathological fractures, spinal cord andnerve compression, bone marrow infiltration, and impaired motility. Inmany cases the systemic presence of a cancer can also make the cancerincurable.

Normal bone is made up of three major cell types: bone-formingosteoblasts, bone-resorbing osteoclasts, and osteocytes. Osteocytes makeup approximately 95% of bone cells and maintain the bone remodelingprocess by coordinating osteolytic and osteoblastic activities. Whencancer cells invade the bone, many of the normal bone functions areaffected. Cancer cells interact with the local microenvironment topromote cancer cell survival via bone destruction and vascularization.

Cx43 hemichannels in osteocytes have been shown to open by treatmentwith alendronate (AD), an efficacious and commonly used bisphosphonatedrug. Bisphosphonates are a class of drugs known for treating many bonedisorders including bone metastasis. Powles et al. have shownadministration of bisphosphonates to be associated with a decrease inthe incidence of bone metastasis and a decrease in death rate inpatients with breast cancer. AD has been associated with decreased tumorgrowth as well as reduced bone destruction and pain. AD inhibitsosteoclast activity and induces the opening of Cx43 hemichannels inosteocytes (Plotkin et al., 2002). However, AD administration isaccompanied by multiple, severe side-effects.

I. Methods Related to the Screening of Drug Candidates as Suppressors ofBone Metastasis

A. In Vitro Assays

Certain embodiments are directed to detection of hemichannel opening invitro using a dye-uptake assay. In certain aspects the dye is afluorescent tracer dye (e.g., ethidium bromide or Lucifer yellow).

In one example of an in vitro assay to detect hemichannel opening afluid flow loop apparatus (FFLA) (Parallel Plate Flow Chamber), ormodification thereof, can be used. One example of an FFLA apparatus isdiagramed in FIG. 1. FFLA mimics dynamic fluid microenvironment in thebone to produce fluid flow shear stress (FFSS). Cells are cultured in aparallel plate flow chamber, exposing the cells to steady laminar fluidflow.

Osteocytes sense mechanical strain produced by FFSS in the osteocytelacuna/canalicular network. It has been proposed that bone fluid flow isdriven by extravascular pressure as well as applied cyclic mechanicalloading of osteocytes and that the peak physiologic loads are 8 to 30dyn/cm². In certain aspects FFSS levels were in range of physiologicalvalues reported from previous studies measuring fluid flow within bone.Fluid shear stress magnitude can be changed by adjusting column heightof the flow loop.

Assays used to assess the functionality of the hemichannels can use afluorescent tracer molecule that is small enough to pass through thepore of the hemichannel. If the hemichannel is closed the moleculescannot pass. If the hemichannel is open the dye can pass through andcause the cell to fluoresce, allowing quantification of thefluorescence. When ethidium bromide attaches to DNA it becomesfluorescent. Lucifer yellow fluoresces once it is located inside of acell.

Dye transfer methods can comprise exposing cells to extracellularfluorescent permeability tracers. Extracellular permeability tracers aremolecules that remain outside of cell unless some condition increasesthe permeability of the cell membrane. In certain aspects the tracershave a mass of less than 1, 2, or 3 kDa. In other aspect the tracer willhave a net charge. Such permeability tracers include, but are notlimited to the anionic dyes Lucifer yellow (LY; net charge=−1) andcationic probes ethidium bromide (Etd; net charge=+1), propidium iodide(PI; net charge=+2). The fluorescence of EtBr is enhanced upon bindingto DNA, increasing the contrast and allowing more easy identification.In certain aspects extracellular dye is removed at different timeperiods or after the application of stimuli to open hemichannels and thefluorescence intensity retained by each cell is quantified. In certainaspects fluorescence intensity is quantified in snap shot images.

FIG. 2 illustrates the results from one example of an in vitro dyetransfer assay. MLO-Y4 osteocytic cells were treated with 20 μM AD for30 min in the absence or presence of 1 μg/ml Cx43(E2) antibody. Ethidiumbromide dye uptake assay was conducted and quantified as compared tonon-treated basal level of uptake. The assays were carried out in thepresence of calcium. Low calcium conditions were used as a control(opens hemichannels). In addition MLO-A5 osteoblasts were treated withAD or AD plus Cx43(E2) antibody were used as negative controls—AD doesnot open Cx43 hemichannels in osteoblasts and opening of osteocytichemichannels induced by AD is blocked by Cx43(E2) antibody.

The materials used in certain aspects of in vitro assays to identifypositive modulators of hemichannels include:

Hemichannel expressing cells or cell lines. Cells or cell linesexpressing the various connexin hemichannels can be obtained, isolated,or engineered using methods and/or expression vectors known in the art.

Osteocytes:

Primary osteocytes isolated from animals (including mouse, rats,rabbits, chicken) etc. or osteocytic cell lines including, but notlimited to MLO-Y4 cells and others.

Cancer Cells:

Breast cancer cell lines: including ER, PR, HER and TP53positive/negative cells (e.g., MD-MBA-231, MCF7, T47D, or ZR751).MDA-MB-231 is mammary gland ductal carcinoma. Py8119 mammary tumor celllines were established from spontaneous mammary tumors arising inC57Bl/6 MMTV-PyMT females (mouse mammary tumor virus promoter-drivenpolyoma middle T transgene) mice. The expression of the oncogene(polyoma middle T transgene) is driven by the Mouse Mammary Tumor Viruspromoter

Prostate cancer cell lines: including androgen receptor and 5α-reductasepositive/negative and androgen sensitive/insensitive cell lines (e.g.,LNCaP-Rf, BM 18, pRNA-1-1/ras, RC58T/hTERT, PPC-1, etc).

Osteoblasts:

MLO-A5 osteoblasts are used as a control because they express Connexin43, but they do not appear to open when stimulated by alendronate.

A “reagent” to be tested includes chemical compounds, peptides,proteins, antisense oligos, and/or microRNA.

Tracer Molecules include, but are not limited to lucifer yellow,ethidium bromide, Evans Blue, Alexa350, Alexa488 and Alexa594.

Cx43(E2):

The Cx43(E2) antibody is specific for Cx43 hemichannels. Cx43E2 bindsthe 2^(nd) extracellular loop of Cx43 hemichannels and preventshemichannel opening.

Methods for determining if a reagent opens hemichannels include one ormore of the following steps:

(a) Isolating, obtaining, or producing a connexin expressing cell orcell line. For example, isolating primary osteocytes from calveria.Other cell types can be isolated using other methods known in the art.In certain aspects calvarial osteocytes are isolated from animals (e.g.,16-day embryonic chicken calvaria or new-born mice). Animals aredecapitated and calvarial bone is dissected and quickly dipped in 70%alcohol. The calvarial bone is then put in αMEM and washed multipletimes with PBS. Cleaned bones are placed in fresh αMEM. The bones areminced and cut into 1.5 mm area size. The bone pieces can be treatedwith collagenase to remove soft tissues and osteoid followed bydecalcification using EDTA. Finally, osteocytes are released from thebone chips by treating with collagenase and vigorous agitation.

(b) Isolating primary osteocytes from long bone. Long bone osteocytescan be isolated from 2-3 week old mice or rats. For example, mice aregiven an overdose of anesthesia, and cervically dislocated, decapitated,and dipped into 70% Ethanol. The femur and tibia with the end of thejoints still intact are isolated. The leg is quickly dipped in 70%alcohol and then placed into αMEM. Legs in αMEM are washed with PBS. Themajor portion of muscle is removed, and detached from thetendons/ligaments. Cleaned bones are placed in fresh αMEM. Once allbones are cleaned, both ends of each bone are cut off using a scalpeljust prior to flushing out the marrow using PBS. Bones are cut into 1.5to 2 mm lengths and treated with collagenase. In one example, the bonepieces are treated with collagenase sequentially 9 times to remove allother tissues and osteoid followed by decalcification using EDTA.

(c) Culturing the cells or cell lines. For example, primary and/orosteocytic cell lines are cultured on collagen-coated plates and arebathed in recording medium (HCO₃-free α-MEM medium buffered with HEPES)containing a permeability tracer.

(d) Administering a test reagent. The cultured cells are placedcontacted with a test reagent for desirable amount of time.

(c) Determining permeability tracer uptake. Permeability tracer uptakeis determined by detecting the amount of tracer inside the cells. Incertain aspects time-lapse recording is used. Fluorescence can berecorded at regions of interest in different cells with an eclipsefilter on a microscope based on the wavelength of the fluorescence ofthe tracer or other probe(s) being used. In certain aspects images arecaptured by fast cooled digital camera every 2 minutes and imageprocessing is performed with ImageJ software. The collected data can beillustrated as fold difference of initial fluorescence and fluorescenceat the time of interest versus the basal fluorescence.

For snapshot images, cells can be exposed to permeability tracer for5-10 minutes, rinsed multiple times with PBS, and fixed withformaldehyde. In certain aspects, at least three microphotographs offluorescence fields are taken with a microscope. Image analysis is donewith ImageJ software. The average of pixel density of random cells ismeasured.

In certain aspects the opening of connexin hemichannels is confirmed.Confirmation can be obtain by, for example, incubating osteocytes withCx43(E2) antibody, a polyclonal antibody specifically inhibiting Cx43hemichannels, along with the test reagent. If the reagent opens Cx43hemichannel, this channel opening will be blocked by Cx43(E2) antibody.To control for the opening of Cx43 hemichannels, osteocytes are treatedwith fluid flow shear stress and/or AD, both known to open hemichannelsin osteocytes.

In a particular example, MLO-Y4 osteocytic cells were treated with 20 μMAD for 30 min in the absence or presence of 1 μg/ml Cx43(E2) antibody.Ethidium bromide dye uptake was conducted and quantified as compared tonon-treated basal level of uptake. The assay was carried out in presenceof calcium. Low calcium conditions can be used as control (openshemichannels). The opening of osteocytic hemichannels induced by AD isblocked by Cx43(E2) antibody.

Opening of Osteocytic Cx43 Hemichannels Mediate a Negative Effect onCancer Cell Migration.

Cx43 hemichannels in osteocytes are opened by administration of AD orFFSS. The opened hemichannels permit the release of various factors intothe medium producing a conditioned medium (CM). The released factor(s)in the AD- or FFSS-treated CM decrease cancer cell migration asdetermined by soft agar and wound healing assays. An example of a softagar assay is diagrammed in FIG. 3. As illustrated in FIG. 4, cancercells treated with control CM exhibit normal migration. The soft agarassay is an assay for anchorage-independent growth, as contrasted withanchorage-dependent growth. Only cancer cells can grow on soft agar andtheir growth on this matrix indicates the extent of the cancer cellproliferation.

In certain aspects the methods include incubating primary osteocytes orosteocyte cell lines with a test reagent in α-MEM for various periods oftime and collecting the supernatant (conditioned media) at various timepoints. In certain aspects breast or prostate cancer cells are incubatedwith CM and cancer cell proliferation, migration, and invasion aredetermined.

Cancer cell growth and viability can be determined using WST-1 (WaterSoluble Tetrazolium salts) assay, viable cell counting using Trypan bluemethod, BrdU DNA incorporation, and cell proliferation assay. For WST-1assay, the cell proliferation is measured at an emission wavelength of450 nm with a Synergy HT Multi-Mode Microplate Reader (Biotek).

Cell migration assays are typically performed in transwell membranefilter inserts in 24-well tissue culture plates (BD Biosciences). Thetranswell membrane filter inserts can be, for example, 6.5-mm diameter,8-μm pore size, and 10-nm thick polycarbonate membranes.

Invasion assays are performed in BD Biocoat Growth Factor ReducedMatrigel Invasion Chambers (BD Biosciences). The cancer cell lines areharvested and resuspended in CM from osteocytes with or without the testreagent. Cancer cell suspensions are added to the upper side of theinserts. Cells are incubated at 37° C. for various periods of time.Cells that do not migrate through the filters are removed, and cellsthat migrate through the inserts are fixed and stained with Hema 3 StatPack (Fisher Scientific). The number of migrated cells in 5 fields ofview per insert is counted under a light microscope.

In certain aspects breast cancer migration is decreased when incubatedin CM from osteocytes treated with AD or FFSS to stimulate Cx43hemichannel opening. When osteocyte Cx43 hemichannels were blocked by E2antibody, this inhibitory effect on cancer cell migration wasattenuated. This decrease in cancer cell migration is not seen whenincubated with CM collected from osteoblasts or when treated directlywith AD. Opening of Cx43 hemichannels is protective against breastcancer cell growth and migration.

B. In Vivo Assays

Other embodiments are directed to detection of connexin hemichannelopening in vivo. One example of an in vivo assay for discovery ofreagents useful in treating cancer metastasis to bone includesdetermining the effect of a candidate reagent on Cx43 hemichannels inosteocytes and on cancer bone metastasis in vivo. In certain aspectsCx43 modulation in osteocytes is determined by injecting candidatereagents into a long bone and using fluorescence tracer dyes (e.g.,calcein or Evans blue) to detect the opening of hemichannels inosteocytes in situ.

Determining the Effect a Compound on the Opening of Hemichannels inOsteocytes in Bone Tissue In Vivo.

One example of an in vivo assay to analyze hemichannels in osteocytesuses 3-4 month old mice or rats. The animals are weighed. A test reagentis introduced into the animal through intraperitoneal (IP) injection.After 2-4 hours, fluorescence tracer dyes (i.e. Evans blue, Alexa 594)are injected into lateral tail vein of the animal or by IP injection.Note: up to 1% of animal's body weight in volume can be injected. Incertain aspects the animal is warmed prior to tail vein injection todilate the tail vein. After 2-4 hours, the animal is scarified andtibial and femur bones free of muscle tissues are dissected and washedmultiple times with PBS. The bone is fixed in paraformaldehyde anddecalcified in 14% EDTA solution at 4° C. for two weeks or roomtemperature under constant agitation for 3-5 days. The bone is washed inPBS and soaked in 30% sucrose in PBS overnight and embedded in OCTcompound. Position of the bone is typically adjusted in the mold asneeded. Five μm thick frozen sections are cut using a cryostat, thesections rinsed in PBS, and mounted using 50% glycerol in PBS. The bonesections can be examined under fluorescence microscope and the degree ofosteocytes in the bone taking up tracer dyes are quantified using ImageJ.

The opening of Cx43 hemichannels in osteocytes can be confirmed bymechanical loading on tibias opening Cx43 hemichannels in osteocytes.This can serve as a positive control for hemichannel opening inosteocytes in vivo. For negative control, mice with the deficiency ofCx43 in osteocytes are used. This mouse is generated by crossing with10-kb DMP-1 Cre and Cx43 flox mice.

The effect of the testing reagent on bone metastasis in vivo isdetermined using an intratibial injection bone metastasis model and/orintracardiac injection cancer metastasis assay.

Intratibial Injection Bone Metastasis Model.

The methods include anesthesizing 1-month old, normal orimmunocompromised mice using isoflurane. The mice are also givenbuprenorphine-HCl (0.3 mg/ml) as an analgesic. Intratibial injectionsare performed using cancer cells expressing fluorescence orchemiluminescence markers (e.g., Py8119 cells expressing Luc-GFP tonormal mice or Luc-GFP-expressing MD-MBA-231 to immunocompromised mice).The cancer cells are inoculated into the bone marrow area of righttibias through a pre-made hole made by a Hamilton syringe fitted with a30-gauge needle. PBS was injected into the left tibias as control. Thetesting reagent or saline is administered IP twice a week for 5 weeks.Intratibial tumor growth is monitored with bioluminescence imaging orfluorescence every week starting from 3 days after tumor cellinoculation. At the termination of the study after sufficientbioluminescence imaging, X-ray images are taken to test bone quality andlabeled metastatic cancer cell colonies are observed and counted with afluorescence microscope.

Intracardiac Injection Bone Metastasis Model.

Two-three month old, normal or immunocompromised mice are anesthetizedby isoflurane and are also given buprenorphine-HCl (0.3 mg/ml) as ananalgesic. Cancer cells expressing fluorescence or chemiluminescencemarkers (e.g., Py8119 cells expressing Luc-GFP to normal mice orLuc-GFP-MD-MBA-231 to immunocompromised mice) are injected into the leftcardiac ventricle of mice. The procedure includes: Holding the needleangled towards the operator and to the right, insert it into the secondintercostal space, approximately 3 mm to the left of the sternum.Advance about 5 mm and turn the needle gently until the pulsatile flowof bright red arterial blood is observed entering the hub. Inject thecell suspension over 30 sec. Withdraw the needle and apply pressure onthe injection site for 30 sec using an alcohol wipe. Place the mouse ona warmed surface until it has fully recovered from anesthesia. Performbioluminescent or fluorescent imaging after intracardiac injection toverify distribution of tumor cells every week from 3 days after tumorcell inoculation. At the termination of the study after sufficientbioluminescence imaging, X-ray images are taken to assess bone qualityand labeled metastatic cancer cell colonies are observed and countedwith a fluorescence microscope.

Cx43 Conditional Knock Out (cKO) Mice.

Because homozygous Cx43 global knockouts are lethal, and also becausethe inventors want to examine the role of Cx43 expressed in osteocytes,osteocyte-specific Cx43 knockout mice were generated. Crossing micehomozygous for the floxed Cx43 gene with Cx43 global heterozygous miceto facilitate the complete deletion of Cx43 in osteocytes. Cx43fl/− mice(50% of progeny) were then crossed with mice expressing Cre recombinasedriven by the human DMP-1 promoter. This created mice that were Cx43fl/−, DMP 1 Cre+ or Cx43 fl/−, DMP 1 Cre− (small percentage areCx43fl/fl or Cx43−/−). Cx43 deficient osteocytes were confirmed byimmunohistochemistry.

Studies can include 4 groups of mice: WT treated with alendronate (AD),KO treated with alendronate, WT without AD, and KO without alendronate.AD was administered to the mice at 150 μg/kg body weight. With ADtreatment it is expected bone metastasis will increase in KO compared toWT mice. And without AD treatment bone metastasis should be similarbetween WT and knockout mice.

II. Methods of Treating Conditions Associated with Connexin Hemichannels

In certain embodiments modulators of connexin hemichannels can be usedto treat disorders associated with connexin hemichannels, includinginflammatory disorders such as osteoarthritis (OA) and spinal injury.The methods and compositions described herein can also be used to treatwounds such as corneal and skin wounds.

A. Osteoarthritis (OA)

Osteoarthritis is a prevalent disease that affects proximate 20% ofadults in the United States. This disease causes the degeneration ofjoints including articular cartilage and subchondral bone. The pathologyof OA is characterized by a loss of articular cartilage leading tonarrowing of joint space, increased joint friction and potentialstructure remodeling. The current treatment includes exercise, lifestylechange and analgesics. If symptoms become severe, joint replacementsurgery is normally performed. Thus far, there is no specificpharmaceutical intervention available for the treatment of OA.

Chondrocytes express connexin (Cx) 43 hemichannels, and these channelsmediate the passage of small molecules (less than 1 kDa) betweeninside/outside of the cell. Under normal condition, Cx43 hemichannels inchondrocytes remain closed; however, these channels are open underinflammatory conditions and release small molecules such aspro-inflammatory factors. The mechanical loading and interleukin-β1induce the opening of Cx43 hemichannels in chondrocytes promoteinflammatory response with the release of inflammatory promoting factorssuch as prostaglandin E2 (PGE2) and ATP.

Inhibiting the opening of Cx43 hemichannels in chondrocytes (e.g., bychemical reagents, etc.), can suppress the inflammation and thedevelopment of osteoarthritis. The hemichannel opening in chondrocytescan be detected using the methods described herein. The release ofpro-inflammatory factors (PGE2 and ATP) by Cx43 hemichannels is measuredusing ELISA assays. Agents that block the opening of hemichannels can beused a therapeutic for inflammatory disorders such as OA.

Elevated interleukin 1β (IL-1β) is an inducer of OA. Abnormal jointloading is also known to increase the risk of OA. The exposure of IL-1βcauses the release of prostaglandins and NO by chondrocytes. Thereleased PGE₂ exerts catabolic effects by inhibiting proteoglycansynthesis and inducing collage degradation. It has been shown thatmechanical loading opens Cx43 hemichannels. Cx43 serves as a portal forthe PGE₂ release in bone cells. Cx43 is expressed on the surface ofchondrocytes (see FIG. 18) and in articular cartilage. IL-1β andmechanical loading caused the opening of Cx43 hemichannels inchondrocytes (FIG. 18) and this opening was inhibited byhemichannel-specific Cx43 antibody. When hemichannels are blocked byCx43(E2) antibody, the inflammatory response evoked by IL-1β wasinhibited.

Based on above evidence, Cx43 hemichannels in chondrocytes are open byIL-1β or mechanical loading, and PGE₂ released by hemichannels leadingto the development of OA. Specific blockade of Cx43 hemichannels inchondrocytes may be used in a therapeutic strategy for the treatment ofOA caused by elevated IL-1β or trauma (abnormal loading).

In vitro cell models for evaluating Cx43 channel activity. Primarychondrocytes are isolated from joints of mouse leg bones. An agent'seffect on Cx43 hemichannel opening and gap junction coupling inchondrocytes can be detected and the time and dosage-dependent effectsevaluated. For example, hemichannel opening is assessed by dye uptakeassay, using Lucifer yellow or Alexa dyes. Downstream effects aremeasured by detecting release of PGE₂ and ATP using ELISA assays.

With spinal injuries, local inflammation and swelling often result fromlocalized injury, trauma, or infection and the same events can also bethe cause of systemic inflammation. Inflammation is often characterizedby increased redness, swelling, temperature, pain, and some loss offunction in the affected area. In certain aspects agents that inhibithemichannels opening can be used to ameliorate inflammation associatedwith central nervous system inflammation and/or spinal cord injury.

Wound healing represents another significant health issue and entails acomplex biological process regardless of causation. In general, thewound is cleaned by infiltrating cells and fluids during the associatedinflammatory response. This initial inflammatory phase is followed by aproliferative phase where different cell types provide the necessaryfactors and tissue environment for wound healing or filling-in byappropriate cells such as fibroblasts, keratinocytes, and a variety ofothers. Additional events such as angiogenesis and contraction of thewound as epithelial cells gradually fill-in the wound also occur. Thisphase tends to last about 7-10 days depending upon the severity of thewound and the efficiency of the inflammatory phase. Circumstances suchas older age, immunodeficiency, as well as stress, and otherenvironmental factors can affect wound healing. Extended exposure of thewound leads to increased possibilities of infection, adverseinflammatory effects, as well as scarring and possibly chronic wounds.Generally, the wound healing process resolves with the maturation andremodeling phase. Collagen is replaced, remodeled, and cross-linked,thereby increasing the strength of the newly developed tissue andunnecessary blood vessels, cells and tissues are slowly removed from thewound site. This final phase can last up to several years as the bodytends to the final healing stage.

Treatments for wounds typically involve the application of antibioticsas well as agents that provide protection from the external environmentsuch as bandages, stitches, second skin, sealants, or other creams andsalves. Additionally, numerous compounds are also available fortreatment of inflammation in the early phase of wound healing, often incombination with steroidal anti-inflammatory compounds orpharmaceuticals. Agents described herein or identified by the methodsdescribed herein can be used to modulate the inflammatory processesassociated with wound healing.

III. Antibodies

Certain aspects of the invention are directed to antibodies thatmodulate, positively or negatively, the function of hemichannels. Anexample of identifying and isolating a monoclonal antibody is describedbelow.

The term “CDR” as used herein refers to a Complementarity DeterminingRegion of an antibody variable domain. Systematic identification ofresidues included in the CDRs have been developed by Kabat et al. (1991,Sequences of Proteins of Immunological Interest, 5th Ed., United StatesPublic Health Service, National Institutes of Health, Bethesda).Variable light chain (VL) CDRs are herein defined to include residues atpositions 27-32 (CDR1), 50-56 (CDR2), and 91-97 (CDR3). Variable heavychain (VH) CDRs are herein defined to include residues at positions27-33 (CDR1), 52-56 (CDR2), and 95-102 (CDR3).

As will be appreciated by those in the art, the CDRs disclosed hereinmay also include variants. Generally, the amino acid identity betweenindividual variant CDRs is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%. Thus, a “variant CDR” is one with the specifiedidentity to the parent CDR of the invention, and shares biologicalfunction, including, but not limited to, at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% of the specificity and/or activity of the parent CDR.

While the site or region for introducing an amino acid sequencevariation is predetermined, the mutation per se need not bepredetermined. For example, in order to optimize the performance of amutation at a given site, random mutagenesis may be conducted at thetarget codon or region and the expressed antigen binding protein CDRvariants screened for the optimal combination of desired activity.Techniques for making substitution mutations at predetermined sites inDNA having a known sequence are well known, for example, M13 primermutagenesis and PCR mutagenesis. Screening of the mutants is done usingassays of antigen binding protein activities as described herein.

Amino acid substitutions are typically of single residues; insertionsusually will be on the order of from about one (1) to about twenty (20)amino acid residues, although considerably larger insertions may betolerated. Deletions range from about one (1) to about twenty (20) aminoacid residues, although in some cases deletions may be much larger.

Substitutions, deletions, insertions or any combination thereof may beused to arrive at a final derivative or variant. Generally these changesare done on a few amino acids to minimize the alteration of themolecule, particularly the immunogenicity and specificity of the antigenbinding protein. However, larger changes may be tolerated in certaincircumstances.

By “Fab” or “Fab region” as used herein is meant the polypeptide thatcomprises the VH, CH1, VL, and CL immunoglobulin domains. Fab may referto this region in isolation, or this region in the context of a fulllength antibody, antibody fragment or Fab fusion protein, or any otherantibody embodiments as outlined herein.

By “Fv” or “Fv fragment” or “Fv region” as used herein is meant apolypeptide that comprises the VL and VH domains of a single antibody.

By “framework” as used herein is meant the region of an antibodyvariable domain exclusive of those regions defined as CDRs. Eachantibody variable domain framework can be further subdivided into thecontiguous regions separated by the CDRs (FR1, FR2, FR3 and FR4).

The term “antigen-binding portion” of an antibody (or simply “antibodyportion”), as used herein, refers to one or more fragments of anantibody that retain the ability to specifically bind to an antigen(e.g., hemichannel). It has been shown that the antigen-binding functionof an antibody can be performed by fragments of a full-length antibody.Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the VLNK, VH, CL and CH1 domains; (ii)a F(ab′)2 fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fab′ fragment,which is essentially an Fab with part of the hinge region (see,FUNDAMENTAL IMMUNOLOGY (Paul ed., 3rd ed. 1993); (iv) a Fd fragmentconsisting of the VH and CH1 domains; (v) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody; (vi) a dAbfragment (Ward et al., (1989) Nature 341:544-546), which consists of aVH domain; (vii) an isolated complementarity determining region (CDR);and (viii) a nanobody, a heavy chain variable region containing a singlevariable domain and two constant domains.

The term “specifically binds” (or “immunospecifically binds”) is notintended to indicate that an antibody binds exclusively to its intendedtarget. Rather, an antibody “specifically binds” if its affinity for itsintended target is about 5-fold greater when compared to its affinityfor a non-target molecule. Suitably there is no significantcross-reaction or cross-binding with undesired substances. The affinityof the antibody will, for example, be at least about 5 fold, such as 10fold, such as 25-fold, especially 50-fold, and particularly 100-fold ormore, greater for a target molecule than its affinity for a non-targetmolecule. In some embodiments, specific binding between an antibody orother binding agent and an antigen means a binding affinity of at least10⁶ M⁻¹. Antibodies may, for example, bind with affinities of at leastabout 10⁷ M⁻¹, such as between about 10⁸ M⁻¹ to about 10⁹ M⁻¹, about 10⁹M⁻¹ to about 10¹⁰ M⁻¹, or about 10¹⁰ M⁻¹ to about 10¹¹ M⁻¹. Antibodiesmay, for example, bind with an EC₅₀ of 50 nM or less, 10 nM or less, 1nM or less, 100 pM or less, or more prcferably 10 pM or less.

Mouse mAb Purification Protocol.

Protein G rather than Protein A is the column of choice for purifyingmouse IgGs, because mouse IgG1 binds much better to Protein G.Supernatants from Hybridoma cultures without fetal calf serum werecollected after 15 days, in order to produce IgG.

In an experiment a GammaBind Plus™ Sepharose Fast flow column was used.The column was cleaned and then equilibrated with binding buffer. About30 ml of buffer solution was used for each of these steps. Dilutedhybridoma supernatant whit binding buffer was then loaded on the column.Fractions of 1.5 ml (elution) were collected. 150 μl of 1 M Tris bufferwith pH 8 was used to neutralize the pH. The column was re-equilibratedwith binding buffer (30-50 ml). Finally 20 μl of sodium azide was addedfor storage. The column binding capacity is 18 mg/ml of mouse IgG. 1ml/min flow rate was used. 50 mM Na phosphate buffer saline with pH 7was used as binding buffer and 0.1 M glycine with pH 2.7 was used aselution buffer. The supernant is mixed 1:1 ratio with the bindingbuffer.

In certain embodiments a mouse monoclonal antibody (M1) was used tostudy functions of connexin Cx43 forming gap junctions or/andhemichannels, where the heavy chain of monoclonal antibody has as aminoacid sequence set forth in SEQ ID NO:2 and the light chain of the ofmonoclonal antibody M1 has an amino acid sequence set forth in SEQ IDNO:4.

In certain embodiments a mouse monoclonal antibody (M2) was used tostudy functions of connexin Cx43 forming gap junctions or/andhemichannels, where the heavy chain of monoclonal antibody has an aminoacid sequence set forth in SEQ ID NO:6 and the light chain of the ofmonoclonal antibody has an amino acid sequence set forth in SEQ ID NO:8.

In certain embodiments a mouse monoclonal antibody (M7) was used tostudy functions of connexin Cx43 forming gap junctions or/andhemichannels, where the heavy chain of monoclonal antibody has an aminoacid sequence set forth in SEQ ID NO: 10 and the light chain of the ofmonoclonal antibody has an amino acid sequence set forth in SEQ ID NO:12.

Immunoblots.

MLO-Y4 Cells were seeded 3×10⁵ at 60 mm dishes for 48 h. Mouse hearttissues were collected in lysis buffer (5 mM Tris, 5 mM EDTA, 5 mM EGTAplus protease inhibitors, 20 l/4 ml phenylmethylsulfonyl fluoride(PMSF), 20 μl/ml N-ethylmaleimide, 10 μl/ml NaVO₄ and 10 μl/mlleupeptin), homogenized and centrifuged at 100,000×g at 4° C. for 30 minand resuspended in lysis buffer. Crude membrane proteins were separatedby 10% SDS-polyacrylamide gel electrophoresis, transferred tonitrocellulose membranes and blotted with anti-Cx43 CT (1:300 dilution)recognizing the C-terminus of Cx43 or anti-Cx43 E2 (1:500 dilution)recognizing the second extracellular loop of Cx43 or the monoclonalantibodies against the second extracellular loop of Cx43 (1:100dilution). Secondary antibodies, infrared IRDye® 800 anti-rabbit IgG(1:15000) (LI-COR, Lincoln, Nebr., USA), fluorescence was detected withOdyssey infrared detection system (LI-COR, Lincoln, Nebr., USA).

Immunofluorescence.

MLO-Y4 cells were cultured on collagen-coated glass coverslip. The cellswere rinsed 2 times with PBS and incubated with cold 70% ethanol for 20min at −20° C. The use of PFA destroys the epitope, which is lysinerich, therefore not recommended. Then the cells were rinsed twice withPBS in order to remove the ethanol. After that the cells were blockedwith blocking solution (2% goat serum, 2% fish skin gelatin, and 1%bovine serum albumin in PBS) overnight. Then, the cells were labeledwith monoclonal antibodies at different concentrations in PBS, followedby FITC-conjugated goat anti-mouse antibody and WGA-alexa594(Invitrogen) (1:400 and 1:1500 in blocking solution respectively). Thecells were observed by Olympus BH-2 fluorescence microscopy and theimages were processed offline with NIH Image J software.

Dye Uptake for Hemichannel Activities.

Dye uptake measurements were evaluated using snap shot photographs.MLO-Y4 cells were plated on the collagen coated 35 mm dish and incubatedwith recording medium, HCO₃ ⁻ free saline medium buffered with 10 mMHEPES salt composition in mM, 154 NaCl, 5.4 KCl, 1.8 CaCl₂, 1.0 MgCl₂, 5Glucose. Medium with low concentration of divalent cation (low[X²]) wasadded 0.5 mM EGTA but not CaCl₂ and MgCl₂. The recording or low[X²]containing 50 μM EtBr for snap shot recording. Cells were exposed to 100μM of EtBr during 5 min, then rinsed 3 times with PBS and fixed with 2%formamide. At least 3 microphotographs of fluorescence fields were takenwith a 10× dry objective in an inverted microscope (Carl Zeiss) with arhodamine filter. The image analysis was made offline with the softwareimage J. The average of pixel density of 30 random cells was measured.

Dye Coupling Assay for Gap Junctions.

MLO-Y4 cells were plated on collagen coated 35 mm dish and incubatedwith recording medium (HCO₃ ⁻ free αMEM medium buffered with 10 mMHEPES). Cells were microinjected using a micromanipulator InjectMan NI 2and Femtojet both from Eppendorf (Eppendorf) at 37° C. with alexafluor350 (Invitrogen, Eugene, Oreg., USA) (10 mM in PBS). Dye transfer wasmeasured after 2 minutes of alexafluor 350 injection. The index of dyecoupling was scored counting the number of cells that were dyetransferred. Dye coupling was observed under an inverted microscopeequipped with Xenon arc lamp illumination and a Nikon eclipse (Nikon,Japan) (excitation wavelengths 330-380 nm; emission wavelengths above420 nm).

Cell Parachute Dye-Transfer Assay for Gap Junctions.

MLO-Y4 cells were grown to confluence in 12 well plates. The donor cellswere incubated with 5 μM calcein red-orange-AM (790 Da) and 5 μM Oregongreen 488 BAPTA-2-AM (1752 Da) for 40 minutes at 37° C. Gap junctionintercellular communication can be followed by simultaneously labelingcells with calcein red-orange as a gap junction permeable tracer dye andthe gap junction channel impermeable dye Oregon green 488 BAPTA-2. Donorcells preloaded were remove form the plate by trypsinization. Preloadedcells were layered (‘parachuted’) over the top of the unlabeledrecipient cells cultured in at a 1:4 donor to receiver ratio. Cells wereallowed to attach for various periods 1 hours, and then carefully washed3 times and fixed in fresh 2% PFA 10 min RT and rinse 3 times again. Thecells were examined with a fluorescence microscope. For calceinred-orange transfer, the threshold was adjusted to clearly distinguishthe dye-transfer boundaries. The dye transfer positive criterion wasdetecting the calcein red-orange/Oregon green 488 BAPTA-2 with contactcells calcein red-orange positive and Oregon green 488 BAPTA-2 negative.The dye transfer was almost undetectable (<1%). The images were taken inplaces where we found Oregon green 488 BAPTA-2 green positive cells.

Fluid Flow Shear Stress to Open Hemichannels.

Fluid flow was created by parallel-plate flow chambers separated by agasket of defined thickness with gravity-driven fluid flow using aperistaltic pump. The thickness of the gasket determined the channelheight, which was adjusted along with flow rate to generate stresslevels of 16 dyn/cm². The circulating medium was α-MEM buffered with 10mM HEPES.

The examples provided as well as the figures are included to demonstratepreferred embodiments of the invention. It should be appreciated bythose of skill in the art that the techniques disclosed in the examplesor figures represent techniques discovered by the inventors to functionwell in the practice of the invention, and thus can be considered toconstitute preferred modes for its practice. However, those of skill inthe art should, in light of the present disclosure, appreciate that manychanges can be made in the specific embodiments which are disclosed andstill obtain a like or similar result without departing from the spiritand scope of the invention.

IV. Pharmaceutical Compositions

Certain aspects include a composition, e.g., a pharmaceuticalcomposition, containing one or a combination of monoclonal antibodies,or antigen-binding portion(s) thereof formulated with a pharmaceuticallyacceptable carrier. Such compositions may include one or a combinationof (e.g., two or more different) antibodies, or immunoconjugatesdescribed herein. For example, a pharmaceutical composition of theinvention can comprise a combination of antibodies that bind todifferent epitopes on the target antigen or that have complementaryactivities.

Pharmaceutical compositions of the invention also can be administered ascombination therapy, i.e., combined with other agents. For example, thecombination therapy can include an anti-hemichannel antibody combinedwith at least one other anti-cancer agent.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forintravenous, intramuscular, subcutaneous, or parenteral administration(e.g., by injection or infusion). Depending on the route ofadministration, the active compound, i.e., antibody, or immunoconjugate,may be coated in a material to protect the compound from the action ofacids and other natural conditions that may inactivate the compound.

Examples of suitable aqueous and nonaqueous carriers that may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositions ofthe invention is contemplated. Supplementary active compounds can alsobe incorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

The amount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thesubject being treated, and the particular mode of administration. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will generally be that amountof the composition which produces a therapeutic effect. Generally, outof one hundred percent, this amount will range from about 0.01 percentto about ninety-nine percent of active ingredient, preferably from about0.1 percent to about 70 percent, most preferably from about 1 percent toabout 30 percent of active ingredient in combination with apharmaceutically acceptable carrier.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

For administration of the antibody, the dosage ranges from about 0.0001to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight.For example dosages can be 0.3 mg/kg body weight, 1 mg/kg body weight, 3mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body weight or withinthe range of 1-10 mg/kg. An exemplary treatment regime entailsadministration once per week, once every two weeks, once every threeweeks, once every four weeks, once a month, once every 3 months or onceevery three to 6 months. Preferred dosage regimens for ananti-hemichannel antibody of the invention include 1 mg/kg body weightor 3 mg/kg body weight via intravenous administration, with the antibodybeing given using one of the following dosing schedules: (i) every fourweeks for six dosages, then every three months; (ii) every three weeks;(iii) 3 mg/kg body weight once followed by 1 mg/kg body weight everythree weeks.

In some methods, two or more monoclonal antibodies with differentbinding specificities are administered simultaneously, in which case thedosage of each antibody administered falls within the ranges indicated.Antibody is usually administered on multiple occasions. Intervalsbetween single dosages can be, for example, weekly, monthly, every threemonths or yearly. Intervals can also be irregular as indicated bymeasuring blood levels of antibody to the target antigen in the patient.In some methods, dosage is adjusted to achieve a plasma antibodyconcentration of about 1-1000 μg/ml and in some methods about 25-300μg/ml.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentinvention employed, the route of administration, the time ofadministration, the rate of excretion of the particular compound beingemployed, the duration of the treatment, other drugs, compounds and/ormaterials used in combination with the particular compositions employed,the age, sex, weight, condition, general health and prior medicalhistory of the patient being treated, and like factors well known in themedical arts.

A “therapeutically effective dosage” of an anti-hemichannel antibodyresults in a decrease in severity of disease symptoms, an increase infrequency and duration of disease symptom-free periods, or a preventionof impairment or disability due to the disease affliction. Atherapeutically effective amount of a therapeutic compound or antibodycan decrease tumor metastasis, or otherwise ameliorate symptoms in asubject. One of ordinary skill in the art would be able to determinesuch amounts based on such factors as the subject's size, the severityof the subject's symptoms, and the particular composition or route ofadministration selected.

A composition of the present invention can be administered via one ormore routes of administration using one or more of a variety of methodsknown in the art. As will be appreciated by the skilled artisan, theroute and/or mode of administration will vary depending upon the desiredresults. Preferred routes of administration for antibodies of theinvention include intravenous, intramuscular, intradermal,intraperitoneal, subcutaneous, or other parenteral routes ofadministration, for example by injection or infusion. The phrase“parenteral administration” as used herein means modes of administrationother than enteral and topical administration, usually by injection, andincludes, without limitation, intravenous, intramuscular, intraarterial,intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular injection and infusion.

1. A monoclonal antibody comprising a heavy chain amino acid sequence ofSEQ ID NO:2, SEQ ID NO:6, or SEQ ID NO: 10; and a light chain amino acidsequence of SEQ ID NO:4.
 2. A method for detecting the presence, levels,and activity of total and cell surface Cx43 hemichannels comprisingcontacting a cell with an antibody comprising a heavy chain amino acidsequence of SEQ ID NO:2, SEQ ID NO:6, or SEQ ID NO:10; and a light chainamino acid sequence of SEQ ID NO:4; and detecting the levels of antibodybound by the cell.
 3. A method for identifying compounds, conditionsand/or treatments that open connexin hemichannels comprising: (a)contacting a cell expressing a connexin hemichannel with a candidatecompound in the presence of a connexin hemichannel permeable dye; (b)measuring the uptake of dye into the cell.
 4. An in vitro method foridentifying a positive regulator of connexin hemichannels comprising:(a) exposing a cultured cell to a reagent or condition that is acandidate positive regulator of connexin hemichannel opening in thepresence of an permeability tracer; (b) determining if the exposure tothe compound or condition increases the uptake of the permeabilitytracer by the cell; and (c) identifying a compound as a positiveregulator of connexin hemichannels if the amount of permeability traceruptake is greater in cells exposed to a reagent or condition as comparedto control cells.
 5. An in situ method for identifying a positiveregulator of connexin hemichannels comprising quantitating the number ofconnexin hemichannels expressing cells having internalized permeabilitytracer in one or more bone sections obtained from an animal that hasbeen treated systemically or intraperitoneally with a candidate reagentand a permeability tracer.
 6. An in vivo method for identifying ananti-metastatic compound comprising; (a) injecting labeled cancer cellsintratibially into a model animal; (b) administering a candidatehemichannel modulating compound to the animal; (c) detecting the labeledcancer cells via imaging; and (d) quantitating the number of labeledcancer cells present in metastatic locations in the animal.
 7. An invivo method of identifying an anti-metastatic compound comprising; (a)injecting labeled cancer cells into a model animal via the leftventricle of the heart; (b) administering a candidate hemichannelsmodulating compound to the animal; (c) imaging the labeled cancer cells;and (d) quantitating labeled cancer cells present in metastaticlocations in the animal.
 8. An in vitro method of using an antibody,compounds or reagents to suppress inflammatory reactions inchondrocytes, comprising administering a hemichannels blocking antibodyto a subject at risk of developing or diagnosed with a pathologicalinflammatory reaction.
 9. An in vivo method of using a monoclonalantibody, compounds or reagents to treat OA, comprising administering ahemichannels blocking antibody to a subject at risk of developing ordiagnosed with osteoarthritis. (a) injecting an antibody, compound orreagent into knee cap cavity. (b) Assessing the inhibition of activationof NF-κB induced by IL-1β (c) Assessing OA development by X-ray,histological analysis and physical movement.